Matrix protein compositions for induction of apoptosis

ABSTRACT

Enamel matrix, enamel matrix derivatives and/or enamel matrix proteins or peptides may be used as therapeutic or prophylactic agents for inducing programmed cell death (apoptosis), in particular in the treatment or prevention of cancer or malignant or benign neoplasms.

FIELD OF INVENTION

The present invention relates to the use of enamel matrix, enamel matrixderivatives and/or enamel matrix proteins or peptides as therapeutic orprophylactic agents for inducing programmed cell death.

BACKGROUND OF THE INVENTION

Enamel matrix proteins such as those present in enamel matrix are bestknown as precursors of dental enamel. Enamel proteins and enamel matrixderivatives have previously been described in the patent literature toinduce hard tissue formation (i.e. enamel formation, cf. U.S. Pat. No.4,672,032 (Slavkin)) or binding between hard tissues (EP-B-0 337 967 andEP-B-0 263 086). In recent animal studies using enamel matrix proteinsfor regeneration of tooth attachment, it has been observed that theregeneration and healing progresses with minimal signs of epithelialinterference. This is in contrast to all other regenerative therapies ofthe periodontium where epithelial downgrowth from the oral cavity intothe lesion is a common complication. To investigate possible restrictiveeffects of enamel matrix proteins on epithelial cell growth, epithelialcells were cultured in the presence of enamel matrix proteins.

SUMMARY OF THE INVENTION

It has surprisingly been found that epithelial cancer cells (HeLa cells)which are not ameloblasts and which are not found in the periodontalenvironment and which do not participate in tooth development undergoapoptosis (programmed or induced cell death) when cultured in thepresence of enamel matrix, enamel matrix derivatives and/or enamelmatrix proteins (in the following collectively termed “active enamelsubstance”). Accordingly, the present invention relates to the use of apreparation of an active enamel substance for the preparation of apharmaceutical composition for the induction of apoptosis.

In another aspect, the invention relates to the use of a preparation ofan active enamel substance for the preparation of a pharmaceuticalcomposition for the prevention or treatment of malignant or benignneoplasms.

In a further aspect, the invention relates to the use of a preparationof an active enamel substance for the preparation of a pharmaceuticalcomposition for the prevention or treatment of cancer.

In a still further aspect, the invention relates to the use of apreparation of an active enamel substance for the preparation of apharmaceutical composition for the (selective) induction of apoptosis inneoplastic cells.

In a still further aspect, the present invention relates to method forinducing apoptosis in neoplastic cells, the method comprising applyingan effective amount of an active enamel substance at or on neoplasticcells.

Apoptosis (programmed or induced cell death) is involved in the focalelimination of certain cells during normal development and in theturnover of cells in healthy adult tissues. Examples where apoptosis hasbeen found to be involved are in organogenesis during embryonic life,e.g. the separation of digits during limb development, cusp and rootformation during tooth development, elimination of worn-out cells in thesmall intestine, and clonal elimination of lymphocytes that mightotherwise react with “self” antigens.

Recent evidence suggests that apoptosis may be equally important in theunderstanding of carcinogenesis and for developing novel therapies fordifferent neoplastic diseases. Thus, it has been shown that apoptosis isinvolved in preventing genome instability from developing in cells inwhich the cell cycle has been perturbed, and it is associated withinjurious stimuli such as therapeutic irradiation and cytotoxic drugsused to treat malignant neoplasms.

Apoptosis is a gene-regulated process involving the synthesis of someproteins that promote apoptosis and others that protect against it. Thepresence of the tumour suppressor gene p53 is responsible for theinitiation of apoptosis as a result of cell injury, in particular injurycaused by DNA double-stranded breaks. In human cancer treatment, this isextremely important because tumour cells from which p53 is absent do notundergo apoptosis when exposed to ionising radiation. In addition, lackof p53 is likely to result in the survival of cells in which DNAmutations have occurred and thus to increase the risk of development ofcancer. Certain cells that have been deprived of growth promotingfactors or whose growth have been arrested with cytotoxic drugs arerendered susceptible to apoptosis by expression of the proto-oncogenec-myc. This gene encodes an essential part of the proliferativemachinery of the cell, and deregulation of the expression of this geneis implicated in most neoplasms. Certain gene products protect cellsfrom apoptosis. Examples of such gene products include those of thebcl-2 gene, the proto-oncogene c-abl and the LMP-1 gene of theEpstein-Barr virus.

A number of different pathways for induction of apoptosis have beendemonstrated. Gamma irradiation acts directly on the chromosomes bycausing DNA strand breaks. Certain hormones such as glucocorticoidsinduce apoptosis in thymocytes, probably via a glucocorticoid receptorcomplex. A third induction pathway is via a direct contact with theplasma membrane of the target cells. An example of this is the effect ofgranzyme B which is released as part of the interaction of cytotoxicT-cells with a target cell.

In epithelial cells, apoptosis has been found to take place when celladhesion to extracellular matrix by means of fibronectin and otherextracellular proteins is blocked. In an immortalised thyroid cell line,it appeared that adhesion, spreading and cytoskeleton organisation weredependent on integrin-fibronectin interaction. It has been shown thatcells become apoptotic when peptides containing the RGD (Arg-Gly-Asp)motif inhibit binding of fibronectin to integrin receptors (cf. M.Vitale et al., J. Clin. Endocrinol. Metab. 83 (10), 1998, pp.3673-3680). Similar findings have been reported for normal cells by,e.g., Z Zhang et al., Proc. Natl. Acad. Sci. USA 92 (13), 1995, pp.6161-6165; H J Kim et al., Am. J. Physiol. 271 (2 pt. 1), 1996, pp.L277-286; G McGill et al., J. Cell Biol. 138 (4), 1997, pp. 901-911.Inhibition of adhesion resulting in cell death not only happens tonormal (non-transformed) cells, but has also been shown for melanomacells. Thus, M D Mason et al., J. R. Soc. Med. 89 (7), 1996, pp.393-395, reported that loss of integrin-mediated signalling inducedapoptosis in melanoma cells within 3 days of treatment with a peptidecontaining the RGD motif blocking the alpha v beta 3 integrin.Furthermore, it has been shown that the RDG motif is anintegrin-recognition motif, and that peptides containing RDG induceapoptosis by activation of caspase-3 which is an enzyme thatparticipates in a cascade resulting in disassembly of the cell (cf. C DBuckley et al., Nature 397, February 1999, pp. 534-539).

Without wishing to be limited to any particular hypothesis, it iscurrently believed that the active enamel substance may exert itsapoptosis inducing effect either by binding to cell surface receptors inan analogous manner to RDG so as to block the site needed for cellularadhesion to extracellular matrix, or by binding to cells surfacestructures such as CD44 or integrins, thereby directly activatingcaspase 3 and triggering the apoptotic pathway described by Buckley etal., supra.

In developing teeth, the enamel is formed by a layer of epithelial cellscalled ameloblasts. These, in turn, are supported by another layer ofepithelial cells providing the ameloblasts with growth factors andnutrients required for their continued existence and function. Theameloblasts synthesize and secrete enamel matrix proteins which,together with mineral and water, consitute the enamel matrix which is anearly developmental stage of dental enamel. Enamel matrix proteins aremainly present during the secretory stage of enamel formation. Aftertheir initial deposition, they are gradually degraded and then lost asenamel development progresses. Also, during enamel development,apoptosis is observed in epithelial cells close to the enamel matrix.This apoptotic event is preceded by translocation of enamel matrixdegradation products from the developing enamel into the enamel organthat is composed of epithelial cells. The active enamel substanceobserved to be useful in the present invention is composed of a numberof proteins and peptides including such degradation products. Theseobservations are also supported by studies of guinea pig molars showingthat processing of enamel proteins is linked to the reduction of thenumber of surrounding epithelial cells (cf. Example 1 below).

DETAILED DESCRIPTION OF THE INVENTION

Apoptosis has attracted considerable interest for the potentialtreatment of cancers and neoplasms. The present inventors surprisinglyobserved that when human epithelial cancer cells (HeLa cells) werecultured in the presence of the active enamel substance they underwentapoptosis (vide Example 2 below). By way of comparison, human connectivetissue cells (fibroblasts) cultured under similar conditions in thepresence of the active enamel substance were stimulated as to growth.Based on these results, the present inventors believe that the activeenamel substance may be used for the (selective) induction of apoptosisin neoplastic cells, specifically in the treatment, e.g. topicaltreatment, of certain types of cancer and benign, semi-malignant (i.e.locally invasive) or malignant neoplasms.

It is currently believed that the active enamel substance may beparticularly beneficial for use in the treatment of epithelially derivedcancers or neoplasms, as the results currently available appear to showthat the active enamel substance exerts its apoptotic effectspecifically on epithelial cells. In the use according to the presentinvention, it is therefore preferred to apply the active enamelsubstance topically at or on affected tissue comprising a substantialproportion of epithelial cells. In particular, such tissue comprisesskin or mucosal tissue. Mucosal tissue which may advantageously betreated with the active enamel substance in accordance with theinvention comprises any tissue which presents a suitable surfaceavailable for topical application of the active enamel substance, eithernaturally or following surgical incision. Examples of such mucosalsurfaces are oral, gastrointestinal, respiratory tract (e.g. lung),cervical or abdominal mucosa.

Other tissues comprising a significant proportion of epithelial cellsare glandular tissues, e.g. mammary gland, pancreas, liver, thyroidgland, bladder, ovary, prostate, sweat gland, salivary gland orpituitary gland tissue.

It has been found, however, that cancer cells derived from other tissuesthan epithelial or mucosal tissue or other tissues comprising asignificant number of epithelial cells also exhibit a marked increase inapoptosis when treated with the preparation of active enamel substanceaccording to the invention (vide Example 3 below). The present inventiontherefore extends to the use of a preparation of the active enamelsubstance in the treatment of cancers or neoplasms in tissue such asbone or muscle tissue.

On surgical removal of a tumour, it is important to reduce the risk thattumour cells migrate from the site of surgery to invade another part ofthe body. In a specific embodiment of the present invention, the activeenamel substance is therefore applied at or on a tumour site before,during or after surgical removal of a tumour or neoplastic tissue tosubstantially reduce the risk of postsurgical metastasis and/or toprevent recurrence of the tumour at the site of surgery. In particular,it is envisaged that the preparation of active enamel substance may beapplied for adjuvant cancer therapy, e.g. in conjunction withconventional radiation therapy. It is currently believed that suchadjuvant therapy using the active enamel substance may both reduce therisk of tumour cell migration (i.e. metastasis) in accordance with thepresent findings and contribute to the healing of wounds often resultingfrom radiation therapy as the active enamel substance has also beenfound to exhibit wound healing properties (vide for instance WO99/43344).

Apart from the treatment of cancer or neoplastic tissue, it iscontemplated that the active enamel substance may be used for theprophylaxis or treatment of warts, in particular warts resulting fromviral infection, e.g. papilloma or condyloma.

Enamel Matrix, Enamel Matrix Derivatives and Enamel Matrix Proteins

Enamel matrix is a precursor to enamel and may be obtained from anyrelevant natural source, i.e. a mammal in which teeth are underdevelopment. A suitable source is developing teeth from slaughteredanimals such as, e.g., calves, pigs or lambs. Another source is forexample fish skin.

Enamel matrix can be prepared from developing teeth as describedpreviously (EP-B-0 337 967 and EP-B-0 263 086). The enamel matrix isscraped off and enamel matrix derivatives are prepared, e.g. byextraction with aqueous solution such as a buffer, a dilute acid or baseor a water/solvent mixture, followed by size exclusion, desalting orother purification steps, optionally followed by freeze-drying. Enzymesmay be deactivated by treatment with heat or solvents, in which case thederivatives may be stored in liquid form without freeze-drying.

In the present context, enamel matrix derivatives are derivatives ofenamel matrix which include one or several of enamel matrix proteins orparts of such proteins, produced naturally by alternate splicing orprocessing, or by either enzymatic or chemical cleavage of a naturallength protein, or by synthesis of polypeptides in vitro or in vivo(recombinant DNA methods or cultivation of diploid cells). Enamel matrixprotein derivatives also include enamel matrix related polypeptides orproteins. The polypeptides or proteins may be bound to a suitablebiodegradable carrier molecule, such as polyamino acids or polysaccharides, or combinations thereof. Furthermore, the term enamelmatrix derivatives also encompasses synthetic analogous substances.

Proteins are biological macromolecules constituted by amino acidresidues linked together by peptide bonds. Proteins, as linear polymersof amino acids, are also called polypeptides. Typically, proteins have50-800 amino acid residues and hence have molecular weights in the rangeof from about 6,000 to about several hundred thousand Daltons or more.Small proteins are called peptides or oligopeptides.

Enamel matrix proteins are proteins which normally are present in enamelmatrix, i.e. the precursor for enamel (Ten Cate: Oral Histology, 1994;Robinson: Eur. J. Oral Science, January 1998, 106 Suppl. 1:282-91), orproteins which can be obtained by cleavage of such proteins. In generalsuch proteins have a molecular weight below 120,000 daltons and includeamelogenins, non-amelogenins, proline-rich non-amelogenins, amelins(ameloblastin, sheathlin) and tuftelins.

Examples of proteins for use according to the invention are amelogenins,proline-rich non-amelogenins, tuftelin, tuft proteins, serum proteins,salivary proteins, amelin, enamelin, ameloblastin, sheathlin, andderivatives thereof, and mixtures thereof. A preparation containing anactive enamel substance for use according to the invention may alsocontain at least two of the aforementioned proteinaceous substances. Acommercial product comprising amelogenins and possibly other enamelmatrix proteins is marketed as EMDOGAIN® (Biora AB).

In general, the major proteins of an enamel matrix are known asamelogenins. They constitute about 90% w/w of the matrix proteins. Theremaining 10% w/w includes proline-rich non-amelogenins, tuftelin, tuftproteins, serum proteins and at least one salivary protein; however,other proteins may also be present such as, e.g., amelin (ameloblastin,sheathlin) which have been identified in association with enamel matrix.Furthermore, the various proteins may be synthesized and/or processed inseveral different sizes (i.e. different molecular weights). Thus, thedominating proteins in enamel matrix, amelogenins, have been found toexist in several different sizes which together form supramolecularaggregates. They are markedly hydrophobic substances which underphysiologically conditions form aggregates. They may carry or becarriers for other proteins or peptides.

Other protein substances are also contemplated to be suitable for useaccording to the present invention. Examples include proteins such asproline-rich proteins and polyproline. Other examples of substanceswhich are contemplated to be suitable for use according to the presentinvention are aggregates of such proteins, of enamel matrix derivativesand/or of enamel matrix proteins as well as metabolites of enamelmatrix, enamel matrix derivatives and enamel matrix proteins. Themetabolites may be of any size ranging from the size of proteins to thatof short peptides.

As mentioned above, the proteins, polypeptides or peptides for useaccording to the invention typically have a molecular weight of at themost about 120 kDa such as, e.g., at the most 100 kDa, 90 kDa, 80 kDa,70 kDa or 60 kDa as determined by SDS Page electrophoresis. As indicatedabove, epithelial cells associated with ameloblasts are believed to beinduced to undergo apoptosis by degradation products migrating from theenamel matrix during dental enamel development. Such degradationproducts, which generally have a molecular weight between about 3 kDaand 25 kDa, such as between 5 kDa and 20 kDa, may be particularlyeffective for use according to the present invention.

The proteins for use according to the invention are normally presentedin the form of a preparation, wherein the protein content of the activeenamel substance in the preparation is in a range of from about 0.05%w/w to 100% w/w such as, e.g., about 5-99% w/w, about 10-95% w/w, about15-90% w/w, about 20-90% w/w, about 30-90% w/w, about 40-85% w/w, about50-80% w/w, about 60-70% w/w, about 70-90% w/w, or about 80-90% w/w.

A preparation of an active enamel substance for use according to theinvention may also contain a mixture of proteins with differentmolecular weights.

The proteins of an enamel matrix can be divided into a high molecularweight part and a low molecular weight part, and it has been found thata well-defined fraction of enamel matrix proteins possesses valuableproperties with respect to treatment of periodontal defects (i.e.periodontal wounds). This fraction contains acetic acid extractableproteins generally referred to as amelogenins and constitutes the lowmolecular weight part of an enamel matrix (cf. EP-B-0 337 967 and EP-B-0263 086).

As discussed above the low molecular weight part of an enamel matrix hasa suitable activity for inducing binding between hard tissues inperiodontal defects. In the present context, however, the activeproteins are not restricted to the low molecular weight part of anenamel matrix. At present, preferred proteins include enamel matrixproteins such as amelogenin, amelin, tuftelin, etc. with molecularweights (as measured in vitro with SDS-PAGE) below about 60,000 daltonsbut proteins having a molecular weight above 60,000 daltons have alsopromising properties as candidates for wound healing, anti-bacterialand/or anti-inflammatory agents.

Accordingly, it is contemplated that the active enamel substance for useaccording to the invention has a molecular weight of up to about 40,000such as, e.g. a molecular weight of between about 5,000 and about25,000.

Within the scope of the present invention are also peptides as describedin WO 97/02730, i.e. peptides which comprise at least one sequenceelement selected from the group consisting of the tetrapeptides DGEA(Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) andDKGE (Asp-Lys-Gly-Glu) and which further comprise an amino acid sequencefrom which a consecutive string of 20 amino acids is identical to adegree of at least 80% with a string of amino acids having the samelength selected from the group consisting of the amino acid sequenceshown in SEQ ID NO:1 and a sequence consisting of amino acids 1 to 103of SEQ ID NO:1 and amino acids 6 to 324 of SEQ ID NO:2 shown in WO97/02730.

By the term “sequence identity” is meant the identity in sequence ofamino acids in the match with respect to identity and position of theamino acids of the peptides. A gap is counted as non-identity for one ormore amino acids as appropriate.

Such peptides may comprise from 6 to 300 amino acids, e.g. at least 20amino acids, at least 30 amino acids, such as at least 60 amino acids,at least 90 amino acids, at least 120 amino acids, at least 150 aminoacids or at least 200 amino acids.

A method for the isolation of enamel matrix proteins involves extractionof the proteins and removal of calcium and phosphate ions fromsolubilized hydroxyapatite by a suitable method, e.g. gel filtration,dialysis or ultrafiltration (see e.g. Janson, J-C & Rydén, L. (Eds.),Protein purification, VCH Publishers 1989 and Harris, ELV & Angal, S.,Protein purification methods—A practical approach, IRL Press, Oxford1990).

A typical lyophilized protein preparation may mainly or exclusively upto 70-90% contain amelogenins with a molecular weight (MW) between40,000 and 5,000 daltons, the 10-30% being made up of smaller peptides,salts and residual water. The main protein bands are at 20 kDa, 12-14kDa and around 5 kDa.

By separating the proteins, e.g. by precipitation, ion-exchangechromatography, preparative electrophoresis, gel permeationchromatography, reversed phase chromatography or affinitychromatography, the different molecular weight amelogenins can bepurified.

The combination of molecular weight amelogenins may be varied, from adominating 20 kDa compound to an aggregate of amelogenins with manydifferent molecular weights between 40 and 5 kDa, and to a dominating 5kDa compound. Other enamel matrix proteins such as amelin, tuftelin orproteolytic enzymes normally found in enamel matrix, can be added andcarried by the amelogenin aggregate.

As an alternative source of the enamel matrix derivatives or proteinsone may also use generally applicable synthetic routes well-known for aperson skilled in the art or use cultivated cells or bacteria modifiedby recombinant DNA-techniques (see, e.g., Sambrook, J. et al.: MolecularCloning, Cold Spring Harbor Laboratory Press, 1989).

Physico-Chemical Properties of Enamel Matrix, Enamel Matrix Derivativesand Enamel Matrix Proteins

In general the enamel matrix, enamel matrix derivatives and enamelmatrix proteins are hydrophobic substances, i.e. less soluble in waterespecially at increased temperatures. In general, these proteins aresoluble at non-physiological pH values and at a low temperature such asabout 4-20° C., while they will aggregate and precipitate at bodytemperature (35-37° C.) and neutral pH.

At least a part of the active enamel substance may be in the form ofaggregates or is capable of forming aggregates after application invivo. The particle size of the aggregates is in a range of from about 20nm to about 1 μm.

It is contemplated that the solubility properties of the active enamelsubstance are of importance in connection with the prophylactic andtherapeutic activity of the substance. When a composition containing theactive enamel substance is administered to e.g. a human, theproteinaceous substances will precipitate due to the pH normallyprevailing under physiological conditions. Thus, a layer of activeenamel substance is formed at the application site and this layer (whichalso may be a molecular layer in those cases where aggregates have beenformed) is difficult to rinse off under physiological conditions.Furthermore, due to the substances bioadhesive properties (see below)the precipitated layer is firmly bound to the tissue also at the marginbetween the precipitated layer and the tissue. The proteinaceous layerthus covers the tissue onto which the active enamel substance orcompositions thereof have been applied and the active enamel substancesare maintained in situ for a prolonged period of time, i.e. it is notnecessary to administer the active enamel substance(s) with shortintervals. Furthermore, the layer formed in situ can almost be comparedto an occlusive dressing, i.e. the layer formed protects the tissue ontowhich the layer is formed from the surroundings.

In order to enable a proteinaceous layer to be formed in situ afterapplication it may be advantageous to incorporate a suitable buffersubstance in a pharmaceutical composition of the active enamelsubstance; the purpose of such a buffer substance could be to avoid thedissolution of the active enamel substance at the application site.

The active enamel substance has also been observed (by the presentinventors) to possess bioadhesive properties, i.e. it has an ability toadhere to skin or mucosal surfaces. These properties are most valuablein connection with a therapeutic and/or prophylactic treatment at leastfor the following reasons:

-   -   the prophylactically and/or therapeutically active substance(s)        can be maintained at the application site for a prolonged period        of time (i.e. i) the administration frequency can be        reduced, ii) a controlled release effect of the active substance        is obtainable and/or iii) a local treatment at the application        site is improved)    -   the active enamel substance may in itself be suitable as a        vehicle for other prophylactically or therapeutically active        substances because a vehicle containing the active enamel        substance can be formulated as a bioadhesive vehicle (i.e. a        novel bioadhesive drug delivery system based on the bioadhesive        properties of the active enamel substance).        Pharmaceutical Compositions

For the administration to an individual (an animal or a human) theactive enamel substance and/or a preparation thereof are preferablyformulated into a pharmaceutical composition containing the activeenamel substance and, optionally, one or more pharmaceuticallyacceptable excipients.

The compositions may be in form of, e.g., solid, semi-solid or fluidcompositions such as, e.g.,

bioabsorbable patches, drenches, dressings, hydrogel dressings,hydrocolloid dressings, films, foams, sheets, bandages, plasters,delivery devices, implants,

powders, granules, granulates, capsules, agarose or chitosan beads,tablets, pills, pellets, microcapsules, microspheres, nanoparticles,

sprays, aerosols, inhalation devices,

gels, hydrogels, pastes, ointments, creams, soaps, suppositories,vagitories,

solutions, dispersions, suspensions, emulsions, mixtures, lotions,enemas,

kits containing e.g. two separate containers, wherein the first one ofthe containers contains the active enamel substance e.g. in powder orfreeze-dried form optionally admixed with other active drug substance(s)and/or pharmaceutically acceptable excipients and the second containercontaining a suitable medium intended to be added to the first containerbefore use in order to obtain a ready-to-use composition;

and in other suitable forms such as, e.g., implants or coating ofimplants or in a form suitable for use in connection with implantationor transplantation.

Compositions for application to the skin or to the mucosa are consideredmost important in connection with the present invention. Thus, acomposition comprising the active enamel substance to be administeredmay be adapted for administration by any suitable route, for example bytopical (dermal), oral, buccal, nasal, aural, rectal or vaginaladministration, or by administration to a body cavity such as, e.g., theoral, gastrointestinal, lung or abdominal cavity. Furthermore, acomposition may be adapted to administration in connection with surgery,e.g. in connection with excision of tumours or neoplastic tissue or inconjunction with radiation therapy.

As mentioned above, a composition of the active enamel substance may besuitable for use during surgery, e.g. for topical application in theform of a gel, film or dry pellet, or as a rinsing solution or treatmentwith a paste or cream on tissue or surfaces.

The compositions may be formulated according to conventionalpharmaceutical practice, see, e.g., “Remington's PharmaceuticalSciences” and “Encyclopedia of Pharmaceutical Technology”, edited bySwarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.

A pharmaceutical composition comprising an active enamel substanceserves as a drug delivery system. In the present context the term “drugdelivery system” denotes a pharmaceutical composition (a pharmaceuticalformulation or a dosage form) which upon administration presents theactive substance to the body of a human or an animal. Thus, the term“drug delivery system” embraces plain pharmaceutical compositions suchas, e.g., creams, ointments, liquids, powders, tablets, etc. as well asmore sophisticated formulations such as sprays, plasters, bandages,dressings, devices, etc.

Apart from the active enamel substance, a pharmaceutical composition foruse according to the invention may comprise pharmaceutically acceptableexcipients.

A pharmaceutically acceptable excipient is a substance which issubstantially harmless to the individual to which the composition is tobe administered. Such an excipient normally fulfils the requirementsgiven by the national health authorities. Official pharmacopoeias suchas e.g. the British Pharmacopoeia, the United States of AmericaPharmacopoeia and The European Pharmacopoeia set standards forpharmaceutically acceptable excipients.

Whether a pharmaceutically acceptable excipient is suitable for use in apharmaceutical composition is generally dependent on which kind ofdosage form is chosen for use for a particular kind of wound. In thefollowing are given examples of suitable pharmaceutically acceptableexcipients for use in different kinds of compositions for use accordingto the invention.

In the following is given a review on relevant pharmaceuticalcompositions for use according to the invention. The review is based onthe particular route of administration. However, it is appreciated thatin those cases where a pharmaceutically acceptable excipient may beemployed in different dosage forms or compositions, the application of aparticular pharmaceutically acceptable excipient is not limited to aparticular dosage form or of a particular function of the excipient.

The choice of pharmaceutically acceptable excipient(s) in a compositionfor use according to the invention and the optimum concentration thereofcannot generally be predicted and must be determined on the basis of anexperimental evaluation of the final composition. However, a personskilled in the art of pharmaceutical formulation can find guidance ine.g., “Remington's Pharmaceutical Sciences”, 18th Edition, MackPublishing Company, Easton, 1990.

Compositions for Injection or Infusion

For systemic, non-topical administration, the composition comprising theactive enamel substance may be in a form suited for systemic injectionor infusion and may, as such, be formulated with sterile water or anisotonic saline or glucose solution. The composition may be sterilisedby conventional sterilisation techniques which are well known in theart. The resulting aqueous solutions may be packaged for use or filteredunder aseptic conditions and lyophilised, the lyophilised preparationbeing combined with the sterile aqueous solution prior toadministration. The composition may contain pharmaceutically acceptableexcipients as required to approximate physiological conditions, such asbuffering agents, tonicity adjusting agents and the like, for instancesodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, etc.

Topical Compositions

For application to the mucosa or the skin, the compositions for useaccording to the invention may contain conventionally non-toxicpharmaceutically acceptable carriers and excipients includingmicrospheres and liposomes.

The compositions for use according to the invention include all kinds ofsolid, semi-solid and fluid compositions. Compositions of particularrelevance are e.g. pastes, ointments, hydrophilic ointments, creams,gels, hydrogels, solutions, emulsions, suspensions, lotions, liniments,shampoos, jellies, soaps, sticks, sprays, powders, films, foams, pads,sponges (e.g. collagen sponges), pads, dressings (such as, e.g.,absorbent wound dressings), drenches, bandages, plasters and transdermaldelivery systems.

The pharmaceutically acceptable excipients may include solvents,buffering agents, preservatives, humectants, chelating agents,antioxidants, stabilizers, emulsifying agents, suspending agents,gel-forming agents, ointment bases, penetration enhancers, perfumes, andskin protective agents.

Examples of solvents are e.g. water, alcohols, vegetable or marine oils(e.g. edible oils like almond oil, castor oil, cacao butter, coconutoil, com oil, cottonseed oil, linseed oil, olive oil, palm oil, peanutoil, poppyseed oil, rapeseed oil, sesame oil, soybean oil, sunfloweroil, and teaseed oil), mineral oils, fatty oils, liquid paraffin,polyethylene glycols, propylene glycols, glycerol, liquidpolyalkylsiloxanes, and mixtures thereof.

Examples of buffering agents are e.g. citric acid, acetic acid, tartaricacid, lactic acid, hydrogenphosphoric acid, diethylamine etc.

Suitable examples of preservatives for use in compositions are parabens,such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben,isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid,benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDMhydantoin, iodopropynyl butylcarbamate, EDTA, benzalconium chloride, andbenzylalcohol, or mixtures of preservatives.

Examples of humectants are glycerin, propylene glycol, sorbitol, lacticacid, urea, and mixtures thereof.

Examples of chelating agents are sodium EDTA and citric acid.

Examples of antioxidants are butylated hydroxy anisole (BHA), ascorbicacid and derivatives thereof, tocopherol and derivatives thereof,cysteine, and mixtures thereof.

Examples of emulsifying agents are naturally occurring gums, e.g. gumacacia or gum tragacanth; naturally occurring phosphatides, e.g. soybeanlecithin; sorbitan monooleate derivatives; wool fats; wool alcohols;sorbitan esters; monoglycerides; fatty alcohols; fatty acid esters (e.g.triglycerides of fatty acids); and mixtures thereof.

Examples of suspending agents are e.g. celluloses and cellulosederivatives such as, e.g., carboxymethyl cellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carraghenan, acacia gum, arabic gum,tragacanth, and mixtures thereof.

Examples of gel bases, viscosity-increasing agents or components whichare able to take up exudate from a wound are: liquid paraffin,polyethylene, fatty oils, colloidal silica or aluminium, zinc soaps,glycerol, propylene glycol, tragacanth, carboxyvinyl polymers,magnesium-aluminium silicates, Carbopol®, hydrophilic polymers such as,e.g. starch or cellulose derivatives such as, e.g.,carboxymethylcellulose, hydroxyethylcellulose and other cellulosederivatives, water-swellable hydrocolloids, carragenans, hyaluronates(e.g. hyaluronate gel optionally containing sodium chloride), andalginates including propylene glycol aginate.

Examples of ointment bases are e.g. beeswax, paraffin, cetanol, cetylpalmitate, vegetable oils, sorbitan esters of fatty acids (Span),polyethylene glycols, and condensation products between sorbitan estersof fatty acids and ethylene oxide, e.g. polyoxyethylene sorbitanmonooleate (Tween).

Examples of hydrophobic or water-emulsifying ointment bases areparaffins, vegetable oils, animal fats, synthetic glycerides, waxes,lanolin, and liquid polyalkylsiloxanes.

Examples of hydrophilic ointment bases are solid macrogols (polyethyleneglycols).

Other examples of ointment bases are triethanolamine soaps, sulphatedfatty alcohol and polysorbates.

Examples of powder components are: alginate, collagen, lactose, powderwhich is able to form a gel when applied to a surgical wound (absorbsliquid/wound exudate). Normally, powders intended for application onlarge open wounds must be sterile and the particles present must bemicronized.

Examples of other excipients are polymers such as carmelose, sodiumcarmelose, hydroxypropylmethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acaciagum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates,cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates andchitosans.

Dressings and/or bandages are also important delivery systems for theactive enamel substance. When dressings are used as dosage form, theactive enamel substance may be admixed with the other ingredients beforeor during the manufacture of the dressing or the active enamel substancemay in some way be coated onto the dressing e.g. by dipping the dressingin a solution or dispersion of the active enamel substance or byspraying a solution or dispersion of the active enamel substance ontothe dressing. Alternatively, the active enamel substance may be appliedin the form of a powder to the dressing. Dressings may be in the form ofabsorbent wound dressings for application to exuding wounds. Dressingsmay also be in the form of hydrogel dressings (e.g. cross-linkedpolymers such as, e.g. Intrasite® which contains carboxymethylcellulose,propylene glycol or polysaccharide, disaccharide and proteins) or in theform of occlusive dressings such as, e.g., alginates, chitosan,hydrophilic polyurethane film, collagen sheets, plates, powders, foams,or sponges, foams (e.g. polyurethane or silicone), hydrocolloids (e.g.carboxymethylcellulose, CMC), collagen and hyaluronic acid-baseddressings including combinations thereof.

The compositions mentioned above for topical administration are mostsuitably for application directly to wounds or they may be suitable forapplication to or for introduction into relevant orifice(s) of the body,e.g. the rectal, urethral, vaginal, aural, nasal or oral orifices. Thecomposition may simply be applied directly on the part to be treatedsuch as, e.g., on the mucosa, or by any convenient route ofadministration.

Compositions which have proved to be of importance in connection withtopical application are those which have thixotropic properties, i.e.the viscosity of the composition is affected e.g. by shaking or stirringso that the viscosity of the composition at the time of administrationcan be reduced and when the composition has been applied, the viscosityincreases so that the composition remains at the application site.

Compositions for Application to Mucosa or Skin

Suitable compositions for use according to the invention may also bepresented in the form of suspensions, emulsions or dispersions. Suchcompositions contains the active enamel substance in admixture with adispersing or wetting agent, suspending agent, and/or one or morepreservatives and other pharmaceutically acceptable excipients. Suchcompositions may also be suitable for use in the delivery of the activeenamel substance to e.g. an intact or damaged mucosa such as the oral,buccal, nasal, rectal, or vaginal mucosa, or for administration tointact or damaged skin, or wounds.

Suitable dispersing or wetting agents are, for example, naturallyoccurring phosphatides, e.g., lecithin, or soybean lecithin;condensation products of ethylene oxide with e.g. a fatty acid, a longchain aliphatic alcohol, or a partial ester derived from fatty acids anda hexitol or a hexitol anhydride, for example polyoxyethylene stearate,polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitanmonooleate, etc.

Suitable suspending agents are, e.g., naturally occurring gums such as,e.g., gum acacia, xanthan gum, or gum tragacanth; celluloses such as,e.g., sodium carboxymethylcellulose, microcrystalline cellulose (e.g.Avicel® RC 591, methylcellulose); alginates and chitosans such as, e.g.,sodium alginate, etc.

Suitable examples of preservatives for use in compositions according tothe invention are the same as those mentioned above.

Rectal and/or Vaginal Compositions

For application to the rectal or vaginal mucosa, suitable compositionsaccording to the invention include suppositories (emulsion or suspensiontype), enemas, and rectal gelatin capsules (solutions or suspensions).Appropriate pharmaceutically acceptable suppository bases include cocoabutter, esterified fatty acids, glycerinated gelatin, and variouswater-soluble or dispersible bases like polyethylene glycols andpolyoxyethylene sorbitan fatty acid esters. Various additives like,e.g., enhancers or surfactants may be incorporated.

Nasal or Pulmonary Compositions

For application to the nasal or pulmonal mucosa (as well as to the oralmucosa), sprays and aerosols for inhalation are suitable compositionsaccording to the invention. In a typical composition, the active enamelsubstance is present in the form of a particulate formulation optionallydispersed in a suitable vehicle. The pharmaceutically acceptablevehicles and excipients and optionally other pharmaceutically acceptablematerials present in the composition such as diluents, enhancers,flavouring agents, preservatives, etc. are all selected in accordancewith conventional pharmaceutical practice in a manner understood by thepersons skilled in the art of formulating pharmaceuticals.

Dosages of Enamel Matrix, Enamel Matrix Derivatives and Enamel MatrixProteins

In a pharmaceutical composition for use according to the invention, anactive enamel substance is generally present in a concentration rangingfrom about 0.01% to about 99.9% w/w. The amount of composition appliedwill normally result in an amount of total protein per cm² area ofaffected tissue corresponding to from about 0.01 mg/cm² to about 20mg/cm² such as from about 0.1 mg/cm² to about 15 mg/cm².

The amount applied of the composition depends on the concentration ofthe active enamel substance in the composition and of the release rateof the active enamel substance from the composition, but is generally ina range corresponding to at the most about 15-20 mg/cm².

In those cases where the active enamel substance is administered in theform of a liquid composition, the concentration of the active enamelsubstance in the composition is in a range corresponding to from about0.1 to about 50 mg/ml. Higher concentrations are in some cases desirableand can also be obtained such as a concentration of at least about 100mg/ml.

The concentration of the active enamel substance in a pharmaceuticalcomposition depends on the specific enamel substance, its potency, theseverity of the disease to be prevented or treated, and the age andcondition of the patient. Methods applicable to selecting relevantconcentrations of the active enamel substance in the pharmaceuticalcomposition are well known to a person skilled in the art and may beperformed according to established guidelines for good clinical practice(GCP) or Investigational New Drug Exemption (“IND”) regulations asdescribed in e.g. International Standard ISO/DIS 14155 Clinicalinvestigation of medical devices, 1994 and ICH (International Committeefor Harmonisation): Harmonised tripartite guideline for good clinicalpractice, Brookwood Medical Publications, Ltd, Surrey, UK, 1996. Aperson skilled in the art would, by use of the methods described instandard textbooks, guidelines and regulations as described above aswell as common general knowledge within the field, be able to select theexact dosage regimen to be implemented for any active enamel substanceand/or selected other active substances and dosage form using merelyroutine experimentation procedures.

In accordance with the present invention, application of the activeenamel substance at or on tumorous tissue may suitably be combined withother forms of tumour treatment, such as surgery, administration ofchemotherapeutic agents and/or radiation therapy of affected tissue.

The invention is further described in the following examples which arenot in any way intended to limit the scope of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described in the following with reference tothe appended drawings, wherein

FIG. 1 is a graph showing the density of human epithelial (HeLa) cellsgrown in the presence and absence of EMD;

FIG. 2 is a graph showing the production of intracellular cAMP of HeLacells grown in the presence or absence of EMD; and

FIG. 3 is a graph showing induced cell death of HeLa cells grown in thepresence of EMD compared to HeLa cells grown in the absence of EMD asmeasured by the level of apoptosis specific nucleic acid degradationproducts.

The present invention is further described in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

Experimental Section

Enamel Matrix Derivative, EMDOGAIN®, from BIORA AB, S-205 12 Malmö,Sweden containing 30 mg freeze-dried enamel matrix protein (in thefollowing abbreviated to EMD) and 1 ml vehicle solution (PropyleneGlycol Alginate), which are mixed prior to application, unless theprotein and the vehicle are tested separately. The weight ratio is about85/5/10 between the main protein peaks at 20, 14 and 5 kDa,respectively.

EXAMPLE 1

Apoptosis in Guinea Pig Dental Epithelium

Tissue Preparation

Two guinea pigs of 200-250 g were anesthetized by carbon dioxide anddecapitated. Bilateral maxilla and mandible were dissected and fixedwith freshly prepared 4% paraformaldehyde in 0.1M phosphate buffer, pH7.4, at 4° C. After fixation, the specimens were decalcified withneutral buffered 10% EDTA, dehydrated with graded ethanol, and embeddedin paraffin. Sections (7 μm) were taken in the following directions:sagittal (mesio-distal) sections to obtain information about the changesin the enamel epithelium in association with the formation of thecementum pearls that are formed on the enamel surface, bucco-lingualsections to obtain information about changes in the epithelial rootsheath that take place when dentin and cementum are formed at the apicalend of the molars, horizontal sections to follow the distribution of theepithelium around the molars. These sections were used forimmunobistochemical demonstration of the epithelial cells and theirbasal lamina as well as the possible occurrence of apoptosis. Somesections were stained with hematoxylin and eosin (H&E).

Immunohistochemical Detection of Laminin and Keratin

The sections were pretreated with 2% hydrogen peroxide to diminishendogenous peroxidase, incubated with 0.1% protease (Sigma, St. Louis,USA) for 15 min, and non-specific reaction was blocked with 4% normalgoat serum (Dako, Copenhagen, Denmark). Then, they were incubated withrabbit anti-laminin antibody (1:1000, Dako, Copenhagen, Denmark) ormouse monoclonal anti-cytokeratin antibody (1:100, Boehringer-Mannheim,Germany) at 4° C. overnight, rinsed with TBS (50 mM Tris buffer saline,pH 7.4), incubated with goat anti-rabbit antibody with HRP (Dako,Copenhagen, Denmark) or goat antimouse antibody with HRP diluted to1:100 (Dako, Copenhagen, Denmark) at room temperature for 60 min. Theimmunoreaction was visualized with 0.1% DAB (diamino benzidine), TBSwere substituted for the primary antibodies in controls TUNEL procedurefor the visualization of apoptosis

The sections were deparaffinized with xylene, rehydrated with gradedethanol, and rinsed with PBS (phosphate-buffered saline, 50 mM sodiumphosphate, 200 mM NaCl, pH 7.4). The sections were incubated withproteinase K (20 μg/ml in PBS, pH 7.4, Sigma, St. Louis, USA) for 15min. at 37° C. to expose the DNA strands, and rinsed with PBS.Endogenous peroxidase was blocked by 2.0% hydrogen peroxide in PBS for 5min. at room temperature, DIG labeled dUTP (deoxyuridine triphosphate)solution and TdT (terminal deoxynucleotidyl transferase) were mixed tomake TUNEL mixture (Oncor, Gaithersburg, Md.). The sections wereincubated with TUNEL mixture for 60 min. at 37° C. DIG labeled dUTPsolution without TdT (terminal deoxynucleotidyl transferase) was used asnegative control. After incubation, the specimens were rinsed with PB Sand reacted with anti-Digoxigenin-peroxidase for 30 min. at roomtemperature. The reaction was visualized by 0.1% DAB with 0-02% hydrogenperoxide in PBS at room temperature. For positive controls, a mandibleand a spleen of rats was observed in the same manner. Apoptotic cellswere distributed in a part of incisal ameloblasts of transition-stageand a part of disintegrated enamel epithelium from FIRS in rat molars.Apoptotic cells were scattered in the spleen of a rat.

Results

The immunohistochemistry with antibodies against keratin showed theepithelial cells in all positions where they could be identified bymeans of ordinary light microscopy. In addition it was possible todistinguish epithelial cells in areas where cells of the enamel organ orthe epithelial root sheath were mingled with mesenchymal cells of thedental follicle. Immunohistochemistry with antibodies against lamininshowed that the epithelial structures were associated with a basementmembrane in some areas and that a basement membrane was missing inothers. The TUNEL method visualized apoptotic bodies in specific regionsof the contiguously growing teeth.

It was observed that ameloblasts underwent apoptosis in early secretorystage, transition stage, maturation stage and reduced enamel epithelialstage. Apoptosis in maturation stage and reduced enamel epithelial stageappeared to be associated with the formation of cementum pearls. At theapical area of the cartilage-like cementum, the enamel organ which had alarge stellate reticulum and lacked the distinct border of outer enamelepithelium was observed. Apoptosis of enamel epithelium was observed inthe same portions as those of cementum pearls. Based on these results,it is considered likely that apoptosis plays an important part in thereduction, transformation, evacuation and migration of enamelepithelium, which is an important step for the formation of cementumduring tooth development.

EXAMPLE 2

Growth of Human Epithelial Cells in the Presence and Absence of EMD

Materials and Methods

Human epithelial cells (HeLa; a human cervical cancer cell line) wereobtained from BioWhittaker, HeLa 07-229c, Lot # 8c2720. The cells weregrown in Modified Eagle's Medium supplemented with 10% fetal calf serum.EMD was supplied by surface coating culture dishes with a 0.1% EMDsolution in 0.1% HAc, and by supplementing the culture medium with 100μg EMD per ml of medium. HeLa cells cultured under similar conditions inthe absence of EMD were used as controls. All experiments were initiatedwith 50,000 cells per ml of culture medium.

Results

(a) HeLa cells were grown in cultures for 24, 48, 72, 96 and 120 hours.Cultures were then washed with PBS and cells were counted in themicroscope using a fixed grid. Five different areas were counted in eachof six parallel cultures at each timepoint. As appears from FIG. 1, HeLacells show a marked decrease in cell density from 48 hours when grown inthe presence of EMD.

(b) HeLa cells were cultured for 24 or 120 hours, washed twice with PBSand centrifuged. 100 μl of cells from each culture (n=6 at eachtimepoint/experiment) were then lysed, and released intracellular cAMPwas measured by competitive enzyme immunoassay (EIA) using an AmershamPharmacia Biotech “Biotrak cAMP EIA” kit (Cat. No. RPN 225) inaccordance with the manufacturer's instructions. Compared to controls,HeLa cells show a marked increase in intracellular cAMP after 24 hoursof growth in the presence of EMD (FIG. 2). This increase could still beobserved after 120 hours in culture. The increase in intracellular CAMPsuggests that cells grown in the presence of EMD generate internalsignal(s) that could be part of pathways for growth regulation anddifferentiation.

(c) HeLa cells were harvested from cultures at 24, 48, 72, 96 or 120hours (n=5 at each timepoint/experiment), washed in PBS and centrifuged.200 μl of cells were lysed, and the level of apoptosis specific nucleicacid degradation products (histone associated DNA fragments) wasquantified by sandwich ELISA using a Boehringer Mannheim “Cell DeathDetection ELISA” kit (Cat. No. 1 774 425) according to themanufacturer's instructions. The results are presented as the ratiobetween EMD treated cells and untreated cells. Hence values above 1indicate induced cell death while values below 1 reflect prolonged cellsurvival. It appears from FIG. 3 that the HeLa cells show a markedincrease in induced cell death when EMD is present in the cultures(values above 1), peaking at 72 hours after addition of EMD.

Based on these results, it is concluded that epithelial cell growth ispoorer in the presence of EMD, and that the presence of EMD in thecultures increased programmed cell death more than two-fold.

EXAMPLE 3

Growth of Human Cancer Cells in the Presence of EMD

Materials and Methods

Human cancer cells were obtained from cell culture banks derived fromtumour tissues from patients undergoing cancer treatment at theNorwegian Cancer Hospital in Oslo, Norway. The cells were grown inDulbecco's Modiefied Eagle's Medium supplemented with 10% fetal calfserum (osteosarcoma cells) or Eagle's Modified Eagle's Mediumsupplemented with 10% fetal calf serum (epithelial derived cells). EMDwas supplied by surface coating culture dishes with a 0.5 mg/ml EMDsolution in 0.01% HAc, and by supplementing the culture medium with 100μg EMD per ml of medium. All experiments were initiated with 50,000cells per ml of culture medium.

Results

Cells were harvested from the cultures at 72 and 120 hours (n=3×3 eachtime). The cells were washed in PBS and centrifuged, and 200 μl of cellsfrom each sample were lysed and the level of apoptosis specific nucleicacid degradation products (histone associated DNA fragments) wasquantified by sandwich ELISA using a Boehringer Mannheim “Cell DeathDetection ELISA” kit (Cat. No. 1 774 425) according to themanufacturer's instructions. The results are presented as the ratiobetween EMD treated cells and untreated cells. Hence values above 1indicate induced cell death while values below 1 reflect prolonged cellsurvival.

It appears from Table 1 below that human cancer cells show a markedincrease (values above 1) in induced cell death in the presence of EMDin the cell cultures, peaking at 72 to 120 hours after addition of EMD.TABLE 1 Tissue of origin 72 hours after 120 Hours after cell lineaddition of EMD addition of EMD Mammary glands MCF-7 1.1 1.3 SK-BR-3 1.11.2 T47D 1.3 1.2 ZR35 2.1 1.8 Osteosarcoma (bone) OHS 2.5 1.4 Melanoma(skin) LOX 1.1 1.1 FEMX-1 1.5 1.4 Ovarian carcinoma OVCAR 3.5 2.5SK-OV-3 2.8 1.4 Rhabdomyo- sarcoma (muscle) RH-28 2.7 11.7

1-29. (Cancelled)
 30. A method of inducing apoptosis in a benign,semi-malignant or malignant neoplasm comprising the step of contactingthe neoplasm with a composition comprising the low molecular weightfraction of acetic acid extractable proteins from an enamel matrix,wherein the low molecular weight fraction of acetic acid extractableproteins from an enamel matrix comprises proteins having a molecularweight of 20 kDa, 14 kDa, 5 kDa as determined by SDS gelelectrophoresis.
 31. The method of claim 30, wherein the enamel matrixis porcine enamel matrix.
 32. The method of claim 30, wherein theneoplasm is an ectodermal neoplasm.
 33. The method of claim 32, whereinthe ectodermal neoplasm is an epithelial neoplasm.
 34. The method ofclaim 30, wherein the neoplasm is a malignancy.
 35. The method of claim34, wherein the malignancy is a human malignancy.
 36. The method ofclaim 34, wherein the malignancy is chosen from breast cancer, cervicalcancer, ovarian cancer, melanoma, osteosarcoma and rhabdosarcoma. 37.The method of claim 30, wherein the composition is administered as apharmaceutical composition.
 38. The method of claim 37, wherein thepharmaceutical composition comprises a pharmaceutically acceptableexcipient.
 39. The method of claim 38, wherein the excipient ispropylene glycol alginate.
 40. The method of claim 38, wherein theexcipient is hyaluronic acid or a salt derivative thereof.
 41. Themethod of claim 30, wherein the composition is administered topically.42. The method of claim 30, wherein the composition is administered inthe range of about 0.1 mg/cm² to about 15 mg/cm².
 43. The method ofclaim 30, wherein the composition is administered in the range of about0.01 mg/cm² to about 20 mg/cm².
 44. The method of claim 30, wherein atleast part of the low molecular weight fraction of acetic acidextractable proteins from an enamel matrix is administered as anaggregate or forms an aggregate after administration.
 45. The method ofclaim 44, wherein the aggregate has a particle size of about 20 nm toabout 1 μm.
 46. The method of claim 30, wherein the 20 kDa, the 14 kDa,and the 5 kDa protein are present in a ratio, based on weight, of85/5/10 respectively.
 47. The method of claim 46, wherein thecomposition is administered as a pharmaceutical composition.
 48. Themethod of claim 47, wherein the pharmaceutical composition comprises apharmaceutically acceptable excipient.
 49. The method of claim 48,wherein the excipient is propylene glycol alginate.
 50. The method ofclaim 48, wherein the excipient is hyaluronic acid or a salt derivativethereof.
 51. The method of claim 46, wherein the composition isadministered in the range of about 0.1 mg/cm² to about 15 mg/cm². 52.The method of claim 46, wherein the composition is administered in therange of about 0.01 mg/cm² to about 20 mg/cm².
 53. The method of claim32, wherein the neoplasm is chosen from a glandular neoplasm, a boneneoplasm, an ovarian neoplasm, a skin neoplasm, a mucosal neoplasm and amuscle neoplasm.